Manuel Maria SIMON (Simon at mail.boku.ac.at) wrote:
:jr at dna.bio.warwick.ac.uk wrote:
: >
: > Dear All,
: >
: >
: ..The problem I have is that after heating the cell extract
: > for 15 min at 65C, the extract "goes cloudy" and the ppt can be spun down. My
: > question is what is this ppt. and will it interefere in the subsequent CAT
: > assay and can I prevent it coming through. ...
: Hello Jaz
: If you´ r doing the assay with TLC separation you might get a smear!
: Therefore I switched to an alternative protocol.
: I´m using 3H Acteyl-Coenzyme A (e.g. Amersham TRK.688 9,25 MBq/ml) and
: cold chloramphenicol. Proteinextracts can be made also by two freeze
: thaw cycles or also by hypotonic swelling. The you extract your reaction
: with Dimethylether. You count the organic phase.
: This is much easier than any protocol I know.
: Contact me if you want to kow details.
: Manuel M. SIMON PhD
: Center of Applied Genetics
: Univ. of BOKU Vienna
: Vienna
: AUSTRIA
If you want to consider alternatives to the usual TLC you should also
read Neumann et al. in BioTechniques Vol.5 No.5 (1987). The authors
describe an assay which allows counting while the reaction is running.
So it's easy to follow the enzyme reaction rather than
just having the end point. Furthermore the assay is really simple -
all in one tube. I use it all the time.
Cheers
Klaus
--
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